Human respiratory syncytial virus (RSV) is an enveloped RNA-containing virus of Family Paramyxoviridae, Order Mononegavirales, the nonsegmented negative strand RNA viruses. RSV is the most important viral agent of pediatric respiratory tract disease worldwide but lacks an approved vaccine or effective antiviral therapy. Other mononegaviruses include measles, mumps, rabies and Ebola viruses, and hence study of the group is of interest in general. We previously showed that the RSV genome is a single negative strand of RNA of 15,222 nucleotides that encodes 10 major mRNAs and 11 proteins. It thus is one of the more complex mononegaviruses. The purpose of this project is to identify the functions of the viral proteins and to reconstruct events in the viral growth cycle under conditions where they can be more readily studied. Part of these studies involves a minireplicon system, in which an short analog of the genome or its antigenome replicative intermediate is expressed intracellularly from a transfected plasmid and complemented by proteins supplied from other plasmids. Another approach involves infectious recombinant virus bearing mutations in relevant genes. Knowledge of the functions of the proteins is important for a basic understanding of this important human pathogen and for the design of live-attenuated recombinant vaccine viruses. One of the RSV proteins, the M2-1 protein, was identified as a novel transcription antitermination protein. Another protein, M2-2, that is expressed from a separate open reading frame from the same mRNA was identified as a novel RNA regulatory protein.